Skin peptides collection and anti-Bd growth inhibition assays

Skin secretions of two L. nigriventer individuals were collected from two sterile polyethylene bags (Whirl-Pak, Nasco) in which they were kept prior to measurement, by washing the bags with ~50 ml of sterile water. Samples were then frozen until further processing. After defrosting, 2 ml of the skin secretion wash was removed for direct testing against Bd. The remaining volume from each sample was acidified to a final volume of 1% HCL to inactivate potential endogenous peptidases (Resnick et al., 1991; Steinborner et al., 1997) and immediately loaded onto C-18 Sep-Pak cartridges (Waters Corporation, Milford, Massachusetts, USA) which were then stored in vials with 2–5 ml of 0.1% HCL until further processing.

We eluted the peptides bound to the Sep-Paks with 70% acetonitrile, 29.9% water, and 0.1% trifluoracetic acid (v/v/v) and centrifuged them under vacuum to concentrate them to dryness. After Sep-Pak purification, we determined the total concentration of the recovered skin peptides by Micro BCA Assay (Pierce, Rockford, Illinois, USA) following manufacturer’s instructions, except that we used bradykinin (RPPGFSPFR; Sigma) to establish a standard curve (Rollins-Smith et al., 2002). Skin peptides were analysed by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI MS) as described in Pask et al. (2012). We analysed each sample by averaging signals from 250 consecutive laser shots. Mass spectrometry data was acquired in the mass/charge (m/z) range 500 to 7,000, truncated at m/z 4,000 and baseline-corrected with Data Explorer v4.4 (Applied Biosystems). The peak values shown represent the monoisotopic mass, [M+H]+. A few signals may show secondary peaks 22 mass units greater than the primary peak and probably represent a peptide plus sodium adduct [M+Na]+. Spectra may also show peaks at m/z 568.1 and 650.0 because of matrix or background signal.

We performed Bd growth inhibition assays with the direct skin secretion solution following the methods explained above, with 50 µl of the direct skin secretion solution being added to the microtiter plate instead of the peptide dilutions. The assays were completed with the isolate Bd VMV 813.